Mycobacterium tuberculosis (Mtb) is adapted to grow in alveolar macrophages that provide an essential niche to establish infection in the host. After phagocytosis, Mtb virulence mechanisms block phagosome maturation permitting intracellular bacillary replication. We previously discovered that attenuated Mtb H37Ra and M. bovis BCG, at low multiplicity of infection (MOI), induce tumor necrosis factor (TNF)-alpha production and prime infected Macrophages for TNF-mediated apoptosis. In contrast, virulent Mtb strains suppress Macrophage apoptosis by interfering with TNF-alpha signals and by upregulating expression of the anti-apoptotic factor MCL- 1. Apoptosis creates conditions unfavorable for Mtb viability and enhances antigen presentation. We postulate that Macrophage apoptosis is an innate host defense against tuberculosis (TB), analogous to the role established for apoptosis of cells infected by viruses. New preliminary studies highlight the complexity of Mtb-induced Macrophage apoptosis. We find that at high MOI, virulent Mtb are equally or even more potent than attenuated strains at inducing Macrophage cell death by an entirely different pathway than occurs after low-MOl challenge. High-MOl Macrophage apoptosis involves a novel TNF- and caspase-independent mechanism triggered by cytosolic translocation of lysosomal proteases. We postulate that high-MOl apoptosis reflects a mechanism for Mtb to exit host Macrophage, analogous to cell death triggered by lytic viruses. In support of this model we found that high-MOl apoptosis progresses rapidly to necrosis, releasing Mtb to the extracellular space without loss of bacillary viability. We propose to investigate the mechanism and regulation of high-MOl apoptosis, to identify Mtb genes that trigger this response, and to explore the implications of high-MOl apoptosis for TB defense. The interaction between Mtb and alveolar Macrophage leading to death of the infected host cell and/or the infecting microbe is a critical issue in TB pathogenesis. A better understanding of the body's defenses against TB is needed to help develop new approaches for the prevention and treatment of this important disease. Our project will produce new knowledge about the fundamental relationship between the microbe that causes TB and the human cell which serves both as its target for infection and as the body's first line of defense against TB disease.